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Sains Malaysiana 54(1)(2025): 151-164
http://doi.org/10.17576/jsm-2025-5401-12
Biological
Control of Aspergillus flavus with Pseudozyma hubeiensis Yeast
from Nutmeg (Myristica fragrans Houtt.)
(Kawalan Biologi Aspergillus flavus dengan Yis Pseudozyma hubeiensis daripada Buah Pala (Myristica
fragrans Houtt.))
DWI N
SUSILOWATI1, CAHYANI HASNA DESWANTI2, NANI RADIASTUTI2,
SRI RAHAJOENINGSIH1, YADI SURYADI1,*,
SUPRIADI3, NURUL HIDAYAH3, SRI WIDAWATI4 &
NINIK SETYOWATI5
1Research Centre for Horticulture-National
Research and Innovation Agency, Jl. Raya Jakarta-Bogor km 46, Cibinong Bogor
16915, West Java Indonesia
Diserahkan: 11 Mac
2024/Diterima: 28 Oktober 2024
Abstract
Yeasts
are potential biocontrol agents for Aspergillus flavus, an
aflatoxin-producing fungus that is present in various agricultural products,
including nutmeg. This study aimed to obtain yeast isolates from nutmeg (seeds,
pulps, and leaves), characterise them, and identify their antagonistic effects
on A. flavus. The antagonistic activities toward A. flavus were
determined by a dual-culture method. Moreover, the possible mechanism
responsible for these antagonistic effects was also analysed. The results
showed that 51 yeast isolates were successfully isolated from nutmeg. The
inhibition percentages of 47.25 ± 1.66% (isolate DP 1341a) and 55.98 ± 1.31%
(isolate DP 1342) were statistically significant (p < 0.05). The
antagonistic mechanisms of the DP 1341a isolate were associated with the
production of volatile organic compounds (32.79 ± 1.01%), a chitinolytic index
(2.51 ± 0.55), and hyperparasitism but not toxin activity. Moreover, the DP
1342 isolate produced volatile organic compounds (54.33 ± 3.13%), exhibited
toxin activity (2.74 ± 0.22) and exhibited hyperparasitism but did not exhibit
chitinase activity. Molecular identification showed that the two yeast isolates
(DP 1341a and DP 1342) were identified as Pseudozyma hubeiensis with
sequence similarity > 99%. Therefore, the selected yeast isolates, P.
hubeiensis DP 1341a and DP 1342, could be further developed as biological
control agents for A. flavus. This finding will also be useful for
improving biological control agents as an eco-friendly and economically viable
disease management strategy.
Keywords:
Antagonist: Aspergillus flavus; Myristica fragrans; Pseudozyma
hubeiensis; yeasts
Abstrak
Yis
merupakan agen kawalan bio yang berpotensi untuk Aspergillus flavus,
iaitu sejenis kulat penghasil aflatoksin, yang hadir dalam pelbagai produk
pertanian termasuk buah pala. Kajian ini bertujuan untuk memperoleh pencilan
yis daripada buah pala (biji, pulpa dan daun), dan kemudian mencirikan serta
mengenal pasti aktiviti antagonis terhadap A. flavus. Aktiviti antagonis
terhadap A. flavus ditentukan dengan kaedah dwi-kultur. Selain itu,
mekanisme yang mungkin bertanggungjawab bagi kesan antagonis juga dianalisis.
Hasil kajian menunjukkan 51 pencilan yis berjaya dipencilkan daripada buah
pala. Peratusan perencatan sebanyak 47.25 ± 1.66% (pencilan DP 1341a) dan 55.98
± 1.31% (pencilan DP 1342) adalah signifikan secara statistik (p <
0.05). Mekanisme antagonis pencilan DP 1341a dikaitkan dengan pengeluaran
sebatian organik meruap (32.79 ± 1.01%), indeks kitinolitik (2.51 ± 0.55),
hiperparasitisme, tetapi tidak menghasilkan aktiviti toksin. Manakala, pencilan
DP 1342 menghasilkan sebatian organik meruap (54.33 ± 3.13%), menunjukkan
aktiviti toksin (2.74 ± 0.22) dan hiperparasit, tetapi tidak menunjukkan
aktiviti kitinase. Pencirian molekul menunjukkan bahawa kedua-dua pencilan yis
tersebut (DP 1341a dan DP 1342) telah dikenal pasti sebagai Pseudozyma
hubeiensis dengan persamaan jujukan > 99%. Oleh itu, pencilan yis P.
hubeiensis DP 1341a dan DP 1342 yang terpilih boleh dibangunkan sebagai agen
kawalan biologi bagi A. flavus. Penemuan ini juga berguna untuk
penambahbaikan agen kawalan biologi sebagai strategi pengurusan penyakit yang
mesra alam dan berdaya maju dari segi ekonomi.
Kata kunci:
Antagonis; Aspergillus flavus; Myristica fragrans; Pseudozyma hubeiensis;
yis
RUJUKAN
Abdel‐Kareem, M.M., Rasmey, A.M.
& Zohri, A.A. 2019. The action mechanism and biocontrol potentiality of
novel isolates of Saccharomyces cerevisiae against the aflatoxigenic Aspergillus
flavus. Letters in Applied Microbiology 68(2): 104-111.
Aiyama, R., Trivittayasil, V. & Tsuta,
M. 2018. Discrimination of aflatoxin contamination level in nutmeg by
fluorescence fingerprint measurement. Food Control 85: 113-118.
Akocak, P.B., Churey, J.J. & Worobo,
R.W. 2015. Antagonistic effect of chitinolytic Pseudomonas and Bacillus on the growth of fungal hyphae and spores of aflatoxigenic Aspergillus
flavus. Food Bioscience 10(1): 48-58.
Allen, T.W., Burpee, L.L. & Buck, J.W.
2004. In vitro attachment of phylloplane yeasts to Botrytis cinerea,
Rhizoctonia solani, and Sclerotinia homoeocarpa. Canadian Journal
of Microbiology 50(12): 1041-1048.
Avis, T.J. & Bélanger, R.R. 2002.
Mechanisms and means of detection of biocontrol activity of Pseudozyma yeasts against plant-pathogenic fungi. FEMS Yeast Research 2(1): 5-8.
Belda, I., Ruiz, J., Alonso, A., Marquina,
D. & Santos, A. 2017. The biology of Pichia membranifaciens killer
toxins. Toxins 9(4): 112.
Campagnollo, F.B., Mousavi, K.A., Borges,
L.L., Bonato, M.A., Fakhri, Y., Barbalho, C.B., Barbalho, R.L., Corassin, C.H.
& Oliveira, C.A. 2020. In vitro and in vivo capacity of
yeast-based products to bind to aflatoxins B1 and M1 in media and foodstuffs: A
systematic review and meta-analysis. Food Research International 137:
109505.
Cao, Z., Xia, W., Zhang, X., Yuan, H.,
Guan, D. & Gao, L. 2020. Hepatotoxicity of nutmeg: A pilot study based on
metabolomics. Biomedicine and Pharmacotherapy 131: 110780.
Citanirmala, N.M.V., Rahayu, W.P. &
Hariyadi, R.W. 2016. Kajian penerapan peraturan Menteri Pertanian Nomor 53 Tahun 2012
untuk pengendalian aflatoksin pada pala. Jurnal Mutu Pangan: Indonesian Journal of Food Quality 3(1): 58-64.
Choińska, R., Piasecka-Jóźwiak,
K., Chabłowska, B., Dumka, J. & Łukaszewicz, A. 2020. Biocontrol
ability and volatile organic compounds production as a putative mode of action
of yeast strains isolated from organic grapes and rye grains. Antonie van
Leeuwenhoek 113(8): 1135-1146.
Contarino, R., Brighina, S., Fallico, B.,
Cirvilleri, G., Parafati, L. & Restuccia, C. 2019. Volatile organic
compounds (VOCs) are produced by biocontrol yeasts. Food Microbiology 82: 70-74.
Dennis, C. & Webster, J. 1971.
Antagonistic properties of species-groups of Trichoderma. II production of
volatile antibiotics. Transactions of the British Mycological Society 57(1): 41-48.
Dharmaputra, O.S., Ambarwati, S.,
Retnowati, I. & Nurfadila, N. 2015. Fungal infection and aflatoxin
contamination in stored nutmeg (Myristica fragrans) kernels at various
stages of delivery chain in North Sulawesi Province. Biotropia 22(2):
129-139.
Dhaslin, Y.F., Issac, R. & Prabha, M.L.
2019. Antioxidant, antimicrobial, and health benefits of nutmeg. Drug
Invention Today 12(1): 167-169.
Directorate General of Plantations. 2021. Statistical
of National Leading Estate Crops Commodity 2019-2021, edited by Gartina,
D. & Sukriya, R.L. pp. 1011.
Dorner, J.W. 2004. Biological control of
aflatoxin contamination of crops. Journal of Toxicology - Toxin Reviews 23(2-3): 425-450.
Farag, M.A., Mohsen, E. & Abd El
Nasser, G. 2018. Sensory metabolites profiling in Myristica fragrans (nutmeg) organs and in response to roasting as analysed via chemometric tools. LWT 97: 684-692.
Farbo, M.G., Urgeghe, P.P., Fiori, S.,
Marcello, A., Oggiano, S., Balmas, V., Hassan, Z.U., Jaoua, S. & Migheli,
Q. 2018. Effect of yeast volatile organic compounds on ochratoxin A-producing Aspergillus
carbonarius and A. ochraceus. International Journal of Food
Microbiology 284: 1-10.
Felsenstein, J. 1985. Confidence limits on
phylogenies: an approach using the bootstrap. Evolution 39(4): 783-791.
Freimstreakr, F.M., Rueda-Mejia, M.P.,
Tilocca, B. & Migheli, Q. 2019. Biocontrol yeasts: Mechanisms and
applications. World Journal Microbiology and Biotechnology 35(10): 1-19.
Golubev, W.I., Pfeiffer, I. & Golubeva,
E.W. 2006. Mycocin production in Pseudozyma tsukubaensis, Mycopathologia 162(4): 313-316.
Gupta, R., Azhar, M. & Kalam, M.A.
2020. An overview of Myristica fragrans (nutmeg) - its benefits and
adverse effects to humans. Indian Journal of Ayurveda and Integrative
Medicine 2(4): 45-50.
Hartati, S., Wiyono, S., Hidayat, S.H.
& Sinaga, M.S. 2023. Potency and mechanism of yeast-like fungus Pseudozyma in controlling anthracnose on chili. Agrosainstek 7(1):
8-16.
Hyun, S.H., Lee, J.G., Park, W.J., Kim,
H.K. & Lee, J.S. 2014. Isolation and diversity of yeasts from fruits and
flowers of orchard in Sinam-Myeon of Yesan-Gun, Chungcheongnam-do, Korea. The
Korean Journal of Mycology 42(1): 21-27.
Ibrahim, M.A., Cantrell, C.L., Jeliazkova,
E.A., Astatkie, T. & Zheljazkov, V.D. 2020. Utilization of nutmeg (Myristica
fragrans Houtt.) seed hydrodistillation time to produce essential oil
fractions with varied compositions and pharmacological effects. Molecules 25(3): 565.
Jaibangyang, S., Nasanit, R. & Limtong,
S. 2021. Effects of temperature and relative humidity on Aflatoxin B1 reduction
in corn grains and antagonistic activities against Aflatoxin-producing Aspergillus
flavus by a volatile organic compound-producing yeast, Kwoniella
heveanensis DMKU-CE82. BioControl 66(3): 433-443.
Jaibangyang, S., Nasanit, R. & Limtong,
S. 2020. Biological control of aflatoxin-producing Aspergillus flavus by
volatile organic compound-producing antagonistic yeasts. BioControl 65(3): 377-386.
Johnson, J.S., Spakowicz, D.J., Hong, B.Y.,
Petersen, L.M., Demkowicz, P., Chen, L., Leopold, S.R., Hanson, B.M., Agresta,
H.O., Gerstein, M., Sodergren, E. & Weinstock, G.M. 2019. Evaluation of 16S
rRNA gene sequencing for species and strain-level microbiome analysis. Nature
Communications 10(1): 5029.
Joubert, P.M. & Doty, S.L. 2018.
Endophytic yeasts: Biology, ecology and applications. In Endophytes of
Forest Trees. Forestry Sciences, edited by Pirttilä, A. & Frank, A.
Springer: Cham.
Kim, S., Lee, H., Lee, S., Lee, J., Ha, J.,
Choi, Y., Yoon, Y. & Choi, K.H. 2017. Microbe-mediated aflatoxin
decontamination of dairy products and feeds. Journal of Dairy Science 100(2): 871-880.
Khunnamwong, P., Lertwattanasakul, N.,
Jindamorakot, S., Suwannarach, N., Matsui, K. & Limtong, S. 2020.
Evaluation of antagonistic activity and mechanisms of endophytic yeasts against
pathogenic fungi causing economic crop diseases. Folia Microbiologica 65(3): 573-590.
Konsue, W., Dethoup, T. & Limtong, S.
2020. Biological control of fruit rot and anthracnose of postharvest mango by
antagonistic yeasts from economic crops leaves. Microorganisms 8(3):
317.
Kurtzman, C.P., Fell, J.W. &. Boekhout,
T. 2011. The Yeasts: A Taxonomic Study. Volume 2. 5th ed. San Diego:
Elsevier. p. 516.
Lima, S.L., Colombo, A.L. & de Almeida
Jr., J.N. 2019. Fungal cell wall: Emerging antifungals and drug resistance. Frontiers
in Microbiology 10: 2573.
Ling, L., Tu, Y., Ma, W., Feng, S., Yang,
C., Zhao, Y., Wang, N., Li, Z., Lu, L. & Zhang, J. 2020. A potentially
important resource: Endophytic yeasts. World Journal of Microbiology and
Biotechnology 36(8): 110.
Liu, G.L., Chi, Z., Wang, G.Y., Wang, Z.P.,
Li, Y. & Chi, Z.M. 2015. Yeast killer toxins, molecular mechanisms of their
action, and their applications. Critical Reviews in Biotechnology 35(2):
222-234.
Mannazzu, I., Domizio, P., Carboni, G.,
Zara, S., Zara, G., Comitini, F., Budroni, M. & Ciani, M. 2019. Yeast
killer toxins: From ecological significance to application. Critical Review
in Biotechnology 39(5): 603-617.
Maryati, D.A. & Ferniah, R.S. 2021.
Molecular and phylogenetic analysis of inulinase-producing yeast isolated from
nira siwalan (Borassus flabellifer) based on ITS sequences. Journal
of Physics: Conference Series 1943(1): 012060.
Mimee, B., Labbe, C. & Bélanger, R.R.
2009. Catabolism of flocculosin, an antimicrobial metabolite produced by Pseudozyma
flocculosa. Glycobiology 19(9): 995-1001.
Montesinos, E. & Bonaterra, A. 2009. Microbial
Pesticides. In Encyclopedia of Microbiology, 3rd ed., edited by
Schaechter, M. Amsterdam: Elsevier. pp. 110-120.
Moradi, M., Rohani, M., Fani, S.R.,
Mosavian, M.T.H., Probst, C. & Khodaygan, P. 2020. Biocontrol potential of
native yeast strains against Aspergillus flavus and aflatoxin production
in pistachio. Food Additives and Contaminants - Part A Chemistry, Analysis,
Control, Exposure and Risk Assessment 37(11): 1963-1973.
Nesci, A.V., Bluma, R.V. & Etcheverry,
M.G. 2005. In vitro selection of maize rhizobacteria to study potential
biological control of Aspergillus section Flavi and aflatoxin
production. European Journal of Plant Pathology 113(2): 159-171.
Palumbo, J.D., Baker, J.L. & Mahoney,
N.E. 2006. Isolation of bacterial antagonists of Aspergillus flavus from
almonds. Microbial Ecology 52(1): 45-52.
Parafati, L., Vitale, A., Restuccia, C.
& Cirvilleri, G. 2015. Biocontrol ability and action mechanism of
food-isolated yeast strains against Botrytis cinerea causing postharvest
bunch rot of table grape. Food Microbiology 47: 85-92.
Pesireron, M., Kaihatu, S., Suneth, R.
& Ayal, Y. 2019. Perbaikan teknik pengendalian hama dan penyakit perkebunan
pala Banda (Myristica fragrans Houtt.) di Maluku. Jurnal Penelitian
Tanaman Industri 25(1): 45-57.
Rahardiyan, D., Poluakan, M. & Moko,
E.M. 2020. Physico-chemical properties of nutmeg (Myristica fragrans Houtt) of North Sulawesi nutmeg. Fullerene
Journal Chemistry 5(1): 23-31.
Safitri, D., Wiyono, S. & Soekarno, B.P.W.
2021. Mode of action of the endophytic yeast Rhodotorula mucilaginosa in
controlling basal stem rot caused by Phytophthora capsici. IOP
Conference Series: Earth and Environmental Science 667(1): 012050.
Saitou, N. & Nei, M. 1987. The
neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular
Biology and Evolution 4(4): 406-425.
Santos, A., Sanchez, A. & Marquira, D.
2004. Yeast as biological agent to control Botrytis cinerae. Microbiological
Research 159(4): 331-339.
Sembiring, B. 2020. Reducing aflatoxin
contamination of nutmeg using drying methods. IOP Conference Series: Earth
and Environmental Science 418(1): 012030.
Sofiana, I., Susilowati, D.N. & Putra,
I.P. 2020. The potential of endophytic fungi as biocontrol and phosphate
solubilization agent in Capsicum anuum. Fungal Territory 3(3):
16-19.
Sukmawati, D., Andrianto, M.H., Arman, Z.,
Ratnaningtyas, N.I., Al Husna, S.N., El-Enshasy, H.A., Dailin, D. & Kenawy,
A.A. 2020. Antagonistic activity of phylloplane yeasts from Moringa oleifera Lam. leaves against Aspergillus flavus UNJCC F-30 from chicken feed. Indian
Phytopathology 73(1): 79-88.
Susilowati, D.N., Rahayuningsih, S.,
Sofiana, I. & Radiastuti, N. 2021. The potential of nutmeg’s microbes (Myristica
fragrans Houtt.) as antagonistic agents against Rigidoporus microporus. Journal of Suboptimal Lands 50(1): 1-13.
Suvarna, S., Dsouza, J., Ragavan, M.L.
& Das, N. 2018. Potential probiotic characterization and effect of
encapsulation of probiotic yeast strains on survival in simulated
gastrointestinal tract condition. Food Science and Biotechnology 27(3):
745-753.
Spadaro, D. & Droby, S. 2016.
Development of biocontrol products for postharvest diseases of fruit: The
importance of elucidating the mechanisms of action of yeast antagonists. Trends
in Food Science and Technology 47(1): 39-49.
Supriadi. 2017. Aflatoxin of nutmeg in
Indonesia and its control. Perspektif 16(2): 102-110.
Syaifudin, M., Jubaedah, D., Yonarta, D.
& Hastuti, Z. 2019. DNA barcoding of snakeskin gourami Trichogaster
pectoralis and blue gourami Trichogaster trichopterus based on
cythocrome c oxidase subunit I (COI) gene. IOP Conference Series: Earth and
Environmental Science 348(1): 012031.
Tayel, A.A., El-Tras, W., Moussa, S.H.
& El-Agamy, M.A. 2013. Antifungal action of Pichia anomala against
aflatoxigenic Aspergillus flavus and its application as a feed
supplement. Society of Chemical Industry 93(13): 3259-3263.
Wang, Q.M., Jia, J.H. & Bai, F.Y. 2006. Pseudozyma hubeiensis sp. nov. and Pseudozyma shanxiensis sp.
nov., novel ustilaginomycetous anamorphic yeast species from plant leaves. International
Journal of Systematic and Evolutionary Microbiology 56(1): 289-293.
White, T.J., Bruns, T., Lee, S.J. &
Taylor, J.W. 1990. Amplification and
direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR
Protocols: A Guide to Methods and Applications, edited by Innis,
M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J. San Diego: Academic Press.
pp. 315-322.
World Health Organization. 2018. Food
Safety Digest. Aflatoxins. Department of Food Safety and Zoonoses Risk.
WHO/NHM/FOS/RAM/18.1
Zajc, J., Gostinčar, C., Černoša,
A. & Gunde-Cimerman, N. 2019. Stress-tolerant yeasts: Opportunistic
pathogenicity versus biocontrol potential. Genes 10(1): 42.
Zang, W.J., Zhai, H.C., Lv, Y.Y., Cai,
J.P., Jia, F., Wang, J.S. & Hu, Y.S. 2019. Expression of a wheat
β-1,3-glucanase in Pichia pastoris and its inhibitory effect on
fungi commonly associated with wheat kernel. Protein Expression and
Purification 154: 134-139.
Zhang, X., Li, B., Zhang, Z., Chen, Y.
& Tian, S. 2020. Antagonistic yeasts: A promising alternative to chemical
fungicides for controlling postharvest decay of fruit. Journal of Fungi 6(3): 158.
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